Side reactions of hydrogen fluoride on peptides containing glutamic acid in or near the C-terminus in solid phase synthesis

Various peptides containing glutamic acid in or near the C-terminus were synthesized by the solid phase method and treated with anhydrous hydrogen fluoride in the presence of anisole. Compounds, apparently resulting from conversion of glutamic acid to a cyclic derivative, and presumably containing anisole, appeared while glutamic acid disappeared.     

Specific reaction of 9-cis-retinoyl fluoride with bovine opsin

Opsin readily undergoes Schiff base formation between an active site lysine and 9-cis- or 11-cis-retinaldehyde to form the visual pigments isorhodopsin (lambda max = 487 nm) and rhodopsin (lambda max = 500 nm), respectively (Dratz, 1977). It would be predicted that 9-cis-retinoyl fluoride (1), an isostere of 9-cis-retinal, should be an active site directed, mechanism-based labeling agent of opsin, since a stable peptide bond should be formed instead of a Schiff base. It is shown here that 9-cis-retinoyl fluoride (1) reacts with opsin in a time-dependent fashion (t1/2 = 9 min at 25 microM 1) to form a new, nonbleachable pigment with a lambda max of approximately 365 nm. beta-Ionone competitively slows down the rate of the reaction. The absorbance of the new pigment at approximately 365 nm is similar to that of model amide compounds. This result is consistent in a general and qualitative way with the Nakanishi-Honig point-charge model for visual pigments which requires that the chromophore be charged, a situation not possible when the retinoid is linked to opsin via a peptide bond rather than a protonated Schiff base [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A., Arnabaldi, M., & Motto, M.G. (1979) J. Am. Chem. Soc. 101, 7084-7086]. 9-cis-Retinoyl fluoride (1) is approximately 4-fold more potent than all-trans-retinoyl fluoride (2) as an inactivator of bovine opsin. Importantly, 13-cis-retinoyl fluoride (3) is inactive, and no new absorption band at 365 nm is observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compositional analysis of polysaccharides via solvolysis with liquid hydrogen fluoride

A simple procedure is described for solvolysis of the glycosidic linkages of polysaccharides and related compounds in and by liquid hydrogen fluoride (HF), which does so without destruction of acid-sensitive sugars or formation of reversion products from the released monosaccharides. Following solvolysis, water is added to effect hydrolysis of the resulting glycosyl fluorides. Liberated reducing sugars are determined qualitatively and quantitatively, without derivatization, by high-performance liquid chromatography.     

Anhydrous hydrogen fluoride deglycosylates glycoproteins.

Exposure of glycoproteins to anhydrous hydrogen fluoride cleaves all the linkages of neutral and acidic sugars within 1 hr at 0°C while leaving peptide bonds and glycopeptide linkages of amino sugars intact. More severe treatment with anhydrous hydrogen fluoride (3 hr at 23°C) cleaves the O-glycosidic linkages of amino sugars, but peptide bonds and the N-glycosidic linkage between asparagine and N-acetylglucosamine still remain intact. Anhydrous hydrogen fluoride, therefore, may be used for the deglycosylation of glycoproteins, thereby assisting in the further purification, proteolysis, and sequencing of the protein component. During the cleavage of glycosidic linkages by anhydrous hydrogen fluoride there is little or no degradation of the sugars themselves, thus allowing their quantitative recovery. Therefore, anhydrous hydrogen fluoride may also be useful in the analysis of complex polysaccharides.     

The reaction of allitol with hydrogen halides

The reaction of allitol with fuming hydrochloric acid at 100° afforded 1,4-anhydro-5,6-dichloro-5,6-dideoxy-DL-talitol (14) and 1,4-anhydro-6-chloro-6-deoxy-DL-allitol (3). 1,4-Anhydro-6-bromo-6-deoxy-DL-allitol (4) and 1,4-anhydro-DL-allitol (6) were obtained from a similar reaction with excess of hydrogen bromide.     

Solid phase reaction of hemoglobin with spillover hydrogen

The reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) between bovine hemoglobin and spillover hydrogen (SH) was studied. It was shown that, in the field of subunit contact, there is a significant decrease in ability for hydrogen exchange by SH. A comparison of the distribution of the isotope label in the hemoglobin α-subunit was carried out for the HSCIE reaction with the hemoglobin complex and with the free α-subunit. To this end, enzymatic hydrolysis of protein under the action of trypsin was carried out. The separation of tritium-labeled tryptic peptides was achieved by HPLC. Changes in availability of polypeptide chain fragments caused by complex formation were calculated using a molecular model. The formation of the protein complex was shown to lead to a decrease in the ability of fragments of α-subunits MFLSFPTTK (A_32?40) and VDPVNFK (A_93?99) for hydrogen replacement by tritium by almost an order of magnitude; hence, their availability to water (1.4 Å) twice decreased on the average. The decrease in ability to an exchange of hydrogen by spillover tritium on the formation of hemoglobin complex was shown to be connected with a reduction in availability of polypeptide chain fragments participating in spatial interactions of subunits with each other. Thus, the HSCIE reaction can be used not only for the preparative obtaining of tritium-labeled compounds, but also for determining the contact area in the formation of protein complexes.     

Effects of diets containing sodium fluoride on mink

Mink (Mustela vison) kits still nursing, and adult male mink were fed diets containing various levels of fluorine (as NaF) to determine the effects on health, growth and pelt quality. Different groups were fed diets containing 25.5 (control), 46.0, 111.5 or 287.0 ppm fluorine (on a wet basis) for 7-8 mo. Gross, radiographic and microscopic changes were seen in bones from some animals ingesting the higher levels of fluorine. Chemical analyses for fluorine generally reflected levels ingested. Fluorine caused no detectable differences in pelt quality. After data were evaluated, tolerance levels in the feed of not more than 50 ppm fluorine for breeding stock and 100 ppm fluorine for animals being raised only for pelts are recommended.     

Problems associated with use of the benzyloxymethyl protecting group for histidines. Formaldehyde adducts formed during cleavage by hydrogen fluoride

The use of N alpha-tert.-butyloxycarbonyl-N pi-benzyloxymethylhistidine in peptide synthesis resulted in significant levels of several different side products attributable to the generation of formaldehyde during the hydrogen fluoride cleavage reaction. Methylated impurities in a decapeptide were isolated and identified. These methylated impurities were attributed to the use of the benzyloxymethyl protecting group for the histidines, since the impurities did not form when the dinitrophenyl protecting group was used. Also, peptides containing benzyloxymethyl-protected histidines in addition to N-terminal cysteines quantitatively yielded their respective N-terminal thiazolidine derivatives upon isolation from standard hydrogen fluoride cleavage mixtures. Thiazolidine ring formation was circumvented by including in the cleavage reaction a formaldehyde scavenger such as cysteine hydrochloride or resorcinol.     

The reaction of Fe(III) myoglobin fluoride with reducing agents.

The reaction of reducing agents with Fe(III) myoglobin fluoride from sperm whale was studied at alkaline pH values. The rate of reduction by dithionite was indistinguishable from the rate of ligand dissociation even when the values of the rate constants for both were only 1.0 X 10(-3)S-1 (at pH 10.6). Reduction by the reduced Methyl Viologen radical ion and reduced Safranine was faster than the rate of dissociation, providing evidence that these reductants can donate electrons to the iron centre via a pathway involving an (undetectable) liganded Fe(II) intermediate.     

The effect of hydrogen fluoride on two pigments in coleus

Coleus blumei Benth. cv. ‘12th Man’ was fumigated with hydrogen fluoride gas. The treatment caused the development of lesions which originally involved the mesophyll but spread to and eventually included the epidermis. An anthocyanin, cyanidin-3,5-diglucoside acylated with p-coumaric acid, was destroyed and it was postulated that the flavanonol, dihydrokaempferol, was converted to the flavone, apigenin. The anthocyanin destruction and pigment conversion occurred following membrane injury and mixing of the cellular constituents.     

Deglycosylation of chondroitin sulfate proteoglycan by hydrogen fluoride in pyridine

The original deglycosylation procedure using HF/pyridine has been modified for maximal removal of carbohydrate from chondroitin sulfate proteoglycan, with minimal alteration of the core protein. Gas-liquid chromatography analysis after treatment for various times showed that 95% of xylose and mannose and 70-85% of other sugars were removed within 30 min, indicating that almost all chondroitin sulfate chains and about 80% of N- and O-linked oligosaccharides were removed. In contrast to the loss of carbohydrate, no change in amino acid composition or loss of immunoreactivity occurred. Longer treatment of up to 16 h resulted in little additional removal of carbohydrate, but did cause a significant decrease in solubility and recovery of the deglycosylated product. Optimal removal of xylose residues after about 1 h was also shown by maximal acceptor activity of the product in a xylosyltransferase assay. Rapid removal of the HF reagent by vacuum evacuation and ion-exchange chromatography, coupled with the reduced time of treatment allowed recovery of an intact, homogenous protein core that is amenable to structural and sequence studies.